International Application No. PCT/US94/05567 (Int'l Publ. No. 94/27137, published Nov. 24, 1994) to the University of Utah Research Foundation discloses an apparatus for multi-analyte homogeneous fluoro-immunoassays. The disclosed apparatus uses a planar waveguide (see, e.g., FIGS. 12A, 12B & 13 of Int'l Publ. No. 94/27137).
Biosensor apparatuses based on optical detection of analytes by fluorescence of tracer molecules, have attracted increasing attention in recent years. Such apparatuses are useful for both diagnostic and research purposes. In particular, biosensors for a solid-phase fluoro-immunoassay, in which an antibody or antibody fragment specific to the desired analyte is immobilized on a substrate, and binding of the analyte to the antibody results either directly or indirectly (for example, by means of a labeled tracer) in a fluorescence signal, are becoming an important class of optical biosensor.
In most solid-phase fluoro-immunoassays, to achieve adequate sensitivity, a "wash" step is required to remove unbound tracer before measuring the fluorescence. This problem is particularly true for detection of analytes present at concentrations below nanomolar, as is the case for many analytes of interest in body fluids including blood, serum and urine. However, the wash step is tedious, and care on the part of the technician is required to produce repeatable and accurate results. This problem was overcome in Int'l Publ. No. 94/27137 by providing a fluoro-immunoassay system in which sensitivity to analyte concentrations of 10.sup.-10 to 10.sup.-13 molar or below is achieved without a wash step.
The optical technique discussed in Int'l Publ. No. 94/27137 is known as total internal reflection (abbreviated "TIR"). Evanescent light is light produced when a light beam traveling in a waveguide is totally internally reflected at the interface between the waveguide and a surrounding medium having a lower refractive index. A portion of the electromagnetic field of the internally reflected light penetrates into the surrounding medium and constitutes the evanescent light field.
The intensity of evanescent light drops off exponentially with distance from the waveguide surface. In a fluoro-immunoassay, evanescent light can be used to selectively excite tracer molecules directly or indirectly bound to an immobilized binding agent, while tracer molecules free in solution beyond the evanescent penetration distance are not excited and thus do not contribute "background" fluorescence. The use of evanescent field properties for fluorescence measurements is sometimes referred to as "evanescent sensing." For a glass or a similar silica-based material, or an optical plastic such as polystyrene, with the surrounding medium being an aqueous solution, the region of effective excitation by evanescent light generally extends about 1000 to 2000 .ANG. (angstroms) from the waveguide surface. This depth is sufficient to excite most of the tracer molecules bound to the capture molecules (antibodies, receptor molecules, and the like, or fragments thereof) on the waveguide surface, without exciting the bulk of the tracer molecules that remain free in solution. The fluorescence thus resulting reflects the amount of tracer bound to the immobilized capture molecules, and in turn the amount of analyte present.
The maximum solution depth for efficient evanescent collection by the waveguide approximates the depth of the region of evanescent penetration into the solution, and thus the waveguide-penetrating portion of the tracer fluorescence can also be used to selectively measure fluorescence from tracer bound to the waveguide surface.
U.S. Pat. Nos. RE 33,064 to Carter, 5,081,012 to Flanagan et al., 4,880,752 to Keck, 5,166,515 to Attridge, and 5,156,976 to Slovacek and Love, and EP publication Nos. 0 517 516 and 0 519 623, both by Slovacek et al., all disclose apparatus for fluoro-immunoassays utilizing evanescent sensing principles.
It is desirable for speed and convenience in "routine" testing, for example testing of blood samples for viral antibodies, to have an evanescent immuno-fluorescent biosensor which is disposable and which provides multi-sample measurement capability. Multi-sample capability would allow at least one test sample, a high control sample (such as a sample pre-loaded with a high concentration of analyte molecules of interest), and a low control sample (such as a blank) to be simultaneously illuminated and measured. Simultaneous multi-sample capability would also speed up the process of analyzing multiple samples and would reduce the effects of variation in the level of exciting light which are known to occur with typical light sources. It is also desirable for a medical practitioner to be able to perform a fluoro-immunoassay in his or her office without having to send the samples to a laboratory.
Thus, a need remains for an evanescent biosensor system which provides the desired sensitivity in a fluoro-immunoassay which can be performed inexpensively and quickly by relatively non-skilled persons.